Cold adapted influenza vaccines (CAV) are live attenuated viruses that infect the upper respiratory tract, including the Nasal Associated Lymphoid Tissue (NALT). Since NALT is a mucosal lymphoid organ, it is presumed to direct the development of B and T cells that most effectively protect the respiratory tract. Despite the potential benefits of CAV influenza vaccines, the use of these vaccines to elicit immunity to zoonotic viruses, such as H5N1 avian influenza, is limited due to the fear of recombination between contemporary influenza strains and the vaccine strain. In addition, CAV influenza vaccines are not approved for children or the elderly. Thus, it is desirable to develop vaccines that, like CAV, can elicit heterotypic immunity and target the NALT and respiratory tract. However, the actual function of NALT is poorly understood and the role of NALT in immune responses to CAV has never been tested. Therefore, the overall goal of this proposal is to specifically evaluate immune responses in NALT elicited by intranasally delivered recombinant protein and cold-adapted influenza virus (CAV). We will determine whether immune responses to CAV or protein antigens are initiated in NALT, whether priming in NALT confers mucosal homing properties on T cells and whether T cells primed in NALT home to the lung tissue and airways. We will also determine whether immune responses to CAV elicit CD4 T cells that are functionally different than those elicited by nasal vaccination with proteins. Moreover, we will determine whether intranasal vaccination with CAV or protein antigens elicit M2e-specific B cells that home to the upper and lower respiratory tract. We will also test how antigens from CAV or recombinant proteins are delivered to lymphocytes in NALT, whether M cells are a part of this process and whether M cells in NALT are similar to those in Peyer's patches NALT. Finally, we will determine whether intranasal immunization with CAV or protein antigens results in long-lived immunity to virulent, heterotypic strains of influenza. Relevance to public health: Influenza A virus is a significant natural pathogen of man that is responsible for an average of 35,000 deaths annually in the US. In addition, particularly virulent influenza viruses periodically emerge and cause millions of deaths in a single season. These emerging strains of influenza, such as the highly virulent H5N1 variant that appeared in Hong Kong in 1997, have the potential to trigger another worldwide pandemic with devastating social and economic consequences. Thus, it is imperative that we develop vaccines that are protective against a wide array of influenza serotypes. This proposal describes experiments that will determine the mechanisms by which intranasal vaccination with cold adapted viruses or purified proteins promote immunity to influenza. This information will be essential for the development of vaccines that promote long-lasting cross-reactive immunity to influenza, a virus that continues to be an emerging infectious disease.